Abstract:
The differentiation of Diplodia pinea from closely related species, such as Diplodia scrobiculata
and Diplodia seriata, and its detection in plant tissue, represented a critical issue for
a long time. Molecular screening tools have recently been developed to address this topic.
In this study we applied one of the most sensitive and rapid diagnostic screening method
so far developed, called High-Resolution Melting Analysis (HRMA), to detect D. pinea in Austrian
pine (Pinus nigra). HRMA exploits differences in the melting behaviour of PCR products
to rapidly identify DNA sequence variants without the need for cumbersome post-PCR
methods. We developed a HRMA method to detect specific fungal sequences in the mitochondrial
small subunit ribosome gene (mt SSU rDNA). The reliability of this technique
was firstly assessed on DNA extracted from pure cultures of D. pinea and closely related
species. Amplicon differences were screened by HRMA and the results confirmed by direct
DNA sequencing. Subsequently, HRMA was tested on DNA from symptomatic and symptomless
pine shoots, and the presence of the fungus was also confirmed by both conventional
and molecular quantitative approaches. The HRMA allowed the distinction of
D. pinea from closely related species, showing specific melting profiles for the each pathogen.
This new molecular technique, here tested in a plantefungus pathosystem for the first
time, was very reliable in both symptomatic and symptomless shoots. HRMA is therefore
a highly effective and accurate technique that permits the rapid screening of pathogens
in the host.
